Method of controlling zoological and aquatic plant growth

ABSTRACT

A method of controlling target aquatic microorganism pest populations by exposing the target population to a toxic amount of an aquacidal compound comprising an anthraquinone. The method is particularly effective for treating ballast water of ships or other enclosed volumes of water subject to transport between or among geographic areas to control the relocation of plants, toxic bacteria, and animals contained in the water.

This application is a continuation-in-part on PCT applicationPCT/US01/05117 filed Feb. 16, 2001,which is a continuation of copendingU.S. patent application Ser. No. 09/506,017 that was filed on Feb. 17,2000 now U.S. Pat. No. 6,340,468, and which claim benefit to U.S.rovisional patent application Ser. No. 60/237,401 that was filed on Oct.4, 2000. The disclosures of these applications are incorporated hereinby reference.

FIELD OF INVENTION

The present invention is directed to a method and compositions forcontrolling aquatic pests, including zoological organisms and plants.More specifically, the invention is directed to a method and compositionfor controlling, inhibiting, and terminating populations of aquatic andmarine pest plants, organisms, and animals in a target treatment zone.The invention is particularly applicable for sterilizing a treated watervolume (whether or not enclosed) of mollusks, dinoflagellates, bacteriaand algae.

BACKGROUND OF THE INVENTION

The discovery in the Summer of 1988 of the Eurasian zebra musselDressiness polymorph in the Great Lakes of North America represents oneof the most significant events in the history of aquatic biologicalinvasion. However, this was not the first event of a non-indigenousspecies entering into U.S. water. Earlier, the spiny water fleaBythotrephes cedarstroemi and the ruffe Gymnocephalus cernuus hadentered the United States from ballast water of European ports. It wassoon discovered that zebra mussel had also entered the U.S. via ballastwater of European origin.

Since the summer of 1988, there have been a number of aquatic speciesthat have entered into the U.S. via ballast water taken from ports ofother countries. It is estimated that several hundred organisms havebeen introduced into the U.S. via ballast water and/or other mechanisms,not limited to fisheries and ocean or coastal currents. As such, theintegrity of the coastal waters of the United States and the Great Lakesbasin has been substantially threatened by the increased rate of aquaticspecies introduction from other countries.

Prior to 1880, various methods for controlling ballast in ships wereused. In fact, many streets in coastal towns are paved with stones onceused for ship ballast. However, shortly before the turn of the century,water as ballast soon replaced these older methods of stabilizing ships.The rate of invasions by non-indigenous aquatic species rosedramatically since the turn of the century, with much of this beingattributed to shipping. As transoceanic travel increased, so to has theinadvertent introduction of non-indigenous species that threaten naturalwaterways. This is a result of the diverse array of organisms that areable to survive the transoceanic travel in ship ballast water, seachests, and on ship hulls. Of these, the ballast water of ships is oneof the primary mechanisms by which organisms have invaded U.S. waters.

Ballast water consists of either fresh or salt water that is pumped intoa vessel to help control its maneuverability as well as trim, stability,and buoyancy. The water used for ballast may be taken at various pointsduring the voyage including the port of departure or destination.Container ships may make as many as 12 port visits/ballast exchangesduring a single round-the-world journey. Any planktonic species orlarvae that is near the ballast intake may be taken up and transportedto the next port of destination. Globally, an estimated 10 billion tonsof ballast water are transferred each year. Each ship may carry from afew hundred gallons (about 2 metric tons) to greater than 100,000 metrictons depending on the size and purpose. More than 640 tons of ballastwater arrive in the coastal waters of the United States every hour.

The risk of invasion through ballast water has risen dramatically in thepast 20 years as a result of larger vessels being used to transportgreater amounts of material into and out of the U.S. It is estimatedthat between 3000-10,000 species of plants and animals are transporteddaily around the world. In regard to those materials being brought intothe U.S., it is of interest to note that materials which containanimals, fruits, vegetables, etc., must be inspected by the U.S.Department of Agriculture in order to satisfy requirements thatpotentially harmful non-indigenous species are excluded. The irony isthat the ship may be able to release ballast water that has beencontaminated with a non-indigenous species. It is through this mechanismthat several hundred species have been introduced into the UnitedStates.

The U.S. Fish and Wildlife Service currently estimates that the annualcost to the North American economy due to the introduction ofnon-indigenous species is more than $100 billion. While ballast wateronly accounts for a minor proportion of these introductions, the coststill runs to tens of billions of dollars in terms of industrialdislocation, clean-up, loss of product and loss of fisheries and othernatural resources.

As noted above, one of the most notorious species introduced in theGreat Lakes of North America is the Eurasian zebra mussel Dreissenapolymorpha, which has become a major threat to inland water suppliesfrom both a recreational and commercial aspect. Unfortunately, theirrange now extends from the Great Lakes to Louisiana and estimatedeconomic losses are estimated at more than $4 billion for the calendaryear 1999. This species is particularly prolific and a reproducingfemale can expel more than 40,000 fertile eggs per season which, uponhatching, may be found in colonies in excess of one hundred thousand persquare meter. Furthermore, the colonies attach themselves to underwaterstructures that include, amongst others, water intake pipes, from whichthey can be readily disseminated into other environments, ship hulls,debris such as discarded automobile tires, sunken ships, and discardedmetal drums. Established colonies often reach a thickness of 20 cm.

Of particular importance is the clogging of water intake pipes by zebramussels that have a devastating industrial effect, especially in suchuses as power plants, where there is a specific need for reliable waterflow rates. Certain power plants have recorded a 50% water flow ratereduction following infestation and, in addition, zebra mussels appearto secrete substances, both in the living and dead state, that causeferrous metal pipes to degrade. An associated problem also occurs inpipes that supply potable water because even following purificationtreatment, the water has an off flavor. This is attributed not only tothe substances released by the living mussels, but especially by thosethat have died and are decaying. The latter most probably producepolyamines, such as cadaverine, which has a particularly obnoxious odorassociated with decaying proteins and is most often noted in decayingmeat.

Other detrimental environmental effects are the result of zebra musselinfestations both directly and indirectly. Of a direct nature are theeffects on phytoplankton. Zebra mussels feed on phytoplankton, which area source of food for fish, especially in lakes and ponds, therebyincreasing the photosynthetic efficiency for other aquatic weed speciesbecause of increased clarity of the water. This has been shown to havedramatic effects on energy flow and food chains in some waters. Somefish species are threatened. The walleye, for example, thrives in cloudywater and it is generally believed by environmentalists that, increasedwater clarity resulted from zebra mussel activity will lead to thedemise of that industry, presently estimated to be $900 million peryear. Large-scale, multi-billion dollar degradations in native GreatLakes fisheries are already being felt as a result of competition fromnon-fishable species such as the Eurasian ruffe (Gymnocephalus cernuus)and the round goby (Proterorhinus marmoratus), which have beenintroduced through ballast water in the last two decades.

As a result of its feeding preferences, zebra mussels may radicallyalter the species composition of the algal community such thatpotentially harmful species may become abundant. An example isMicrocystis, a blue-green alga of little nutritive value and capable ofproducing toxins which can cause gastrointestinal problems in humans.There are records of Microcystis blooms in Lake Erie and adjacentwaterways. Toxic dinoflagellates such as Prorocentrum, Gymnodinium,Alexandrium and Gonyaulax often appear as blooms, sometimes known as“red tides”, in many parts of the world. Apart from causing serious(sometimes fatal) ailments in several vertebrate consumers, includinghumans, several of these organisms have had devastating effects onshellfish industries in several countries and it is now accepted thatballast-water introductions were responsible in many of these cases.

Reports of the introduction of the cholera bacterium, Vibrio cholera, tothe Gulf coast of the United States have now been traced to theimportation of this species associated with planktonic copepod(crustacean) vectors in ballast water arriving at Gulf coast ports fromSouth America. This, in turn, had been transported from Europe to SouthAmerican ports by similar means.

As a result of the introduction of non-indigenous species into theUnited States, and in order to reduce the possibility of theintroduction of other organisms in the future, in 1990 the U.S. Congresspassed an act known as Public Law 101-646 “The Nonindigenous AquaticNuisance Prevention and Control Act” under the “National Ballast WaterControl Program” which it mandates, among other things, studies in thecontrol of the introduction of aquatic pests into the U.S. These controlmeasures may include UV irradiation, filtration, altering watersalinity, mechanical agitation, ultrasonic treatment, ozonation, thermaltreatment, electrical treatment, oxygen deprivation, and chemicaltreatment as potential methods to control the introduction of aquaticpests. It is likely that other governments will pass similar legislationin the near future as the scope and costs of aquatic pest contaminationbecome better understood.

Numerous methods and compositions have been proposed to control andinhibit the growth of various marine plants and animals. In particular,a number of compositions have been proposed to treat water and varioussurfaces having infestation of zebra mussels. Examples of variouscompositions are disclosed in U.S. Pat. Nos. 5,851,408, 5,160,047,5,900,157 and 5,851,408. Treatment of various aquatic pests, other thantoxic bacteria, is described in WO 00/56140 using juglone or analogsthereof.

These prior compositions and methods, although somewhat effective, havenot been able to completely control the introduction of marine plantsand animals into waterways. Accordingly there is a continuing need inthe industry for the improved control of aquatic pests in the form ofplants and animals, preferably aquatic flora, fauna, and other organismsthat can be suspended in water and are susceptible to geographicmigration by water intake, currents, or tides.

SUMMARY OF THE INVENTION

The present invention is directed to a method of controlling aquaticpests in the form of plants, animals, bacteria, or other microorganisms.The invention is particularly well suited for population control andsterilization of mollusks, dinoflagellates, toxic bacteria, and algae.One aspect of the invention is directed to a method and composition fortreating water to sterilize the treated water of small and micro-sizedaquatic pests including plants, animals, toxic bacteria, andmicroorganisms.

An object of the invention is to provide a method of treating water in adesignated region of open water, an enclosed or a flow-restricted regionto sterilize the area of aquatic pest microorganisms including plants,toxic bacteria, suspended animals, and other biologic organisms insedimentary materials using at least one aquacidally active compound inan effective amount to be toxic to the target species.

A further object of the invention is to provide a method of treatingballast water in ships to control the transport of mollusks,dinoflagellates, toxic bacteria, algae and other microorganisms bytreating the ballast water with an effective amount of an aquacidalcompound to sterilize the ballast water.

Another object of the invention is to provide a method of treating waterat an intake pipe of a process water system to sterilize the water ofplants, animals and microorganisms.

A further object of the invention is to provide a method of treatingballast water to kill aquatic organisms found therein and to controltheir spread.

Still another object of the invention is to provide a method of treatinga volume of water in an enclosed space or localized region of open waterwith a toxic amount of an aquacidal compound which is readily degradedto nontoxic by-products.

Another object of the invention to provide a method of inhibiting thespread of aquatic pests such as adult zebra mussels, zebra mussellarvae, oyster larvae, algal phytoplankton Isochrysis galbana,Neochloris, chlorella, toxic dinoflagellates (e.g. Prorocentrum), marineand freshwater protozoans and toxic bacteria (including vegetativecultures and encysted forms thereof), adult and larval copepods (vectorsof Vibrio Cholera and Vibrio fischeri) and other planktonic crustaceans,e.g., Artemia salina, fish larvae and eggs by treating the water with anamount of at least one aquacidal compound of the type described hereinin a quantity and for a sufficient period of time to kill the targetaquatic pests.

A further object of the invention is to provide aquacidal compounds forthe treatment of ballast water and water in other enclosed spaces, asbiocidal additives to marine paints, and as agrochemicals for applyingto plants for controlling snails and slugs.

Still another object of the invention is to provide a method of treatingwaste water from industrial and municipal sources to kill or control thespread of aquatic pest plant, animal and microorganisms.

These and other objects of the invention that will become apparent fromthe description herein are attained by method of inhibiting the growthof and preferably killing a population of a target pest microorganism byexposing said population to an effective amount of at least oneaquacidal compound selected from the group consisting of: (a) quinones,(b) anthraquinones, (c) quinine, (d) warfarin, (e) coumarins, (f)amphotalide, (g) cyclohexadiene-1,4-dione, (h) phenidione, (i) pirdone,(j) sodium rhodizonate, (j) apirulosin, (k) thymoquinone, and (l)naphthalenediones which have the chemical structure of:

wherein:

R₁ is hydrogen, hydroxy or methyl group;

R₂ is hydrogen, methyl, sodium bisulfate, chloro, acetonyl,3-methyl-2-butenyl, hydroxy, or 2-oxypropyl group;

R₃ is hydrogen, methyl, chloro, methoxy, or 3-methyl-2-butenyl group;

R₄ is hydrogen or methoxy group;

R₅ is hydrogen, hydroxy or methyl group;

R₆ is hydrogen or hydroxy group.

The aquacidal compounds according to the present invention aresurprisingly effective in controlling populations of aquatic pestorganisms at very low concentrations. Typical target aquatic pests smalland microorganisms that are translocated by movement of the surroundingwater, e.g., currents, tides, and intake ports. When the aquacides ofthe invention allowed to remain in contact with the target pestorganisms for a period within the range of several hours to severaldays, the target pest population is killed. The aquacidal compounds arethen degraded through the effects of ultraviolet light, oxidation,hydrolysis, and other natural mechanisms into benign by-products thatallow the treated water to be returned to beneficial use.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is generally directed to a method of treatingwater that hosts a target population of aquatic pests with an aquacidalagent for a sufficient period of exposure to reduce the targetpopulation in the treated water to benign levels or sterilize thetreated water of the target population. The treated water can be locatedin a localized open water region, enclosed space or in a restricted flowpath. Exemplary bodies of water that can be treated according to theinvention include ship ballast water reservoirs, commercial processwater taken in from a static or dynamic body of water, water ready to bedischarged into a holding reservoir or waterway, cooling or other formsof holding ponds, intakes ports or pipes, discharge ports or pipes, heatexchangers, sewage treatment systems, food and beverage processingplants, pulp and paper mills, power plant intake and outlet pipes,cooling canals, water softening plants, sewage effluent, evaporativecondensers, air wash water, canary and food processing water, brewerypasteurizing water, and the like. It is envisioned that the aquacidalagents of the present invention can also be used to treat shore areas orswimming regions if an aquatic pest population has reduced therecreational value of a region of water in a localized or localizablearea in an otherwise open body of water.

In its preferred embodiments, the aquacidal agent made of one or moreaquacidal compounds is added to ship ballast water at a concentrationand for a period of exposure to the aquacidal compound that is effectivein sterilizing the ballast water of target pests microorganisms. Suchconcentrations are typically sufficiently low to become diluted to anon-toxic level when discharged to a larger body of water so as to avoidor minimize harm to the indigenous species of plants and animals. Such atreatment method should help to prevent unintended migration of pestmicroorganisms between and among ports without significant capitalexpense or significant changes in commercial shipping practice.

The aquacidal compounds of the invention are mixed into the water usingstandard dispensing devices and dispensing methods as known in the art.The aquacidal compound can be dispensed as a single dose or over aperiod of time to maintain a desired concentration. Preferably, theaquacidal compound is introduced at a turbulent zone or other area whereagitation will mix the aquacidal compound throughout the water to betreated. The aquacidal compound can be fed intermittently, continuously,or in one batch.

Target Pest Populations

Aquatic pest organisms and populations that can be controlled, killed,or otherwise rendered benign by the method of the invention aregenerally not free ranging between geographical regions of their ownefforts but are subject primarily to the movement of the water currentsor sediment around them. Such microorganisms move primarily under theinfluence of currents, tides, and ballast water taken in at one port anddischarged at another. Aquatic pest microorganisms and populations thatare targets for treatment according to the present invention includebacteria, viruses, protists, fungi, molds, aquatic pest plants, aquaticpest animals, parasites, pathogens, and symbionts of any of theseorganisms. A more specific list of aquatic pest organisms that can betreated according to the invention include, but are not limited to thefollowing categories (which may overlap in some instances):

1) Holoplanktonic organisms such as phytoplankton (diatoms,dinoflagellates, blue-green algae, nanoplankton, and picoplankton) andzooplankton (jellyfish, comb jellies, hydrozoan, polychaete worms,rotifers, planktonic gastropods, snails, copedods, isopods, mysids,krill, arrow worms, and pelagic tunicates), and fish.

2) Meroplanktonic Organisms such as Phytoplankton (propagules of benthicplants) and Zooplankton (larvae of benthic invertebrates such assponges, sea anemones, corals, mollusks, mussels, clams, oysters, andscallops).

3) Demersal organisms such as small crustaceans.

4) Tychoplanktonic organisms such as flatworms, polychaetes, insectlarvae, mites and nematodes.

5) Benthic organisms such as leaches, insect larvae and adults.

6) Floating, Detached Biota such as sea grass, sea weed, and marshplants.

7) Fish and shellfish diseases, pathogens, and parasites.

8) Bythotrephes cederstroemi (spiny water flea, spiny tailed waterflea).

9) Macroinvertebrates, such as mollusks, crustaceans, sponges, annelids,bryozoans and tunicates. Examples of mollusks that can be effectivelycontrolled are mussels, such as zebra mussels, clams, including asiaticclams, oysters and snails.

In further embodiments, the animals being treated are selected from thegroup consisting of bacteria, e.g., Vibrio spp. (V. Cholera and V.Fischeri), Cyanobacteria (blue-green algae), protozoans, e.g.Crytosporidiurn, Giardia, Naeglaria, algae, e.g., Pyrrophyta(dinoflagellates, e.g. Gymnodinium, Alexandrium, Pfiesteria, GonyaulaxGlenodinium (including encysted forms)), Cryptophyta, Chrysophyta,Porifera (sponges), Platyhelminthes (flat-worms, e.g., Trematoda,Cestoda, Turbellaria), Pseudocoelomates (e.g., Rotifers, Nematodes),Annelid worms (e.g., polychaetes, oligochates), Mollusks (e.g.,Gastropods, such as polmonate snails), Bivalves, e.g., Crassostrea(oysters), Mytilus (blue mussels), Dreissena (zebra mussels),Crustaceans, larval-adult forms of copepods, ostracods, mysids,gammarids, larval forms of decapods, and Larval teleost fish.

The method of the invention in a first embodiment adds an effectiveamount of at least one marine plant and animal growth inhibitingcompound to the water to be treated. The aquacidal compound is selectedfrom the group consisting of a quinone, naphthalenedione, anthraquinone,and mixtures thereof. The quinones have the formula:

where R₁ is hydrogen, methyl, hydroxy or methoxy group;

R₂ is hydrogen, hydroxy, methyl, methoxy or —NO₂ group;

R₃ is hydrogen, hydroxy, methyl or methoxy group; and

R₄ is hydrogen, methyl, methoxy, hydroxy, or —NO₂ group.

Examples of quinones found to be effective in controlling or inhibitingplant and animal growth in water include 1,4,benzoquinone (quinone),2,5-dihydroxy 3,6-dinitro-p-benzoquinone (nitranilic acid),2,6-dimethoxybenzoquinone, 3-hydroxy-2-methoxy-5-methyl-p-benzoquinone(fumagatin), 2-methylbenzoquinone (toluquinone),tetrahydroxy-p-benzoquinone (tetraquinone),2,3-methoxy-5-methyl-1,4-benzoquinone,2,3-methoxy-5-methyl-1,4-benzoquinone, and mixtures thereof. In furtherembodiments, the quinone can be an ubiquinone having the formula

where n is an integer from 1 to 12. A particularly preferred ubiquinonehas the formula above where n=10. In further embodiments, the ubiquinonehas the above formula where n=6 to 10 and n is an integer.

In the embodiments where the marine plant and animal inhibitingcomposition is a naphthalenedione other than juglone, suchnaphthalenediones having the formula:

wherein R₁ is hydrogen, hydroxy or methyl group;

R₂ is hydrogen, methyl, sodium bisulfate, chloro, acetonyl,3-methyl-2-butenyl or 2-oxypropyl group;

R₃ is hydrogen, methyl, chloro, methoxy, or 3-methyl-2-butenyl group;

R₄ is hydrogen or methoxy group;

R₅ is hydrogen, hydroxy or methyl group;

R₆ is hydrogen or hydroxy group.

Examples of naphthalenediones include 1,4-naphthalenedione,2-methyl-5-hydroxy-1,4-naphthalenedione (plumbagin),2-methyl-1,4-naphthalenedione (Vitamin K₃), 2-methyl-2 sodiummetabisulfite-1,4-naphthalenedione, 6,8-dihydroxy benzoquinone,2,7-dimethyl-1-4-naphthalenedione (chimaphilia),2,3-dichloro-1,4-naphthalenedione (dichlorine),3-acetonyl-5,8-dihydroxy-6-methoxy-1,4-naphthalenedione (javanicin),2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthalenedione (lapachol),pirdone, and 2-hydroxy-3-methyl-1,4-naphthalenedione (phthiocol).

The anthraquinones have the formula:

wherein R₁ is hydrogen, hydroxy or chloro;

R₂ is hydrogen, methyl, chloro, hydroxy, carbonyl, or carboxyl group;

R₃ is hydrogen or methyl group;

R₄ is hydrogen;

R₅ is hydrogen or hydroxyl group;

R₆ and R₇ are hydrogen; and

R₈ is hydrogen or hydroxyl group.

Examples of anthraquinones that are suitable for treating water tocontrol or inhibit marine plant and animal growth include 9,10anthraquinone, 1,2-dihydroxyanthraquinone (alizarin),3-methyl-1,8-dihydroxyanthraquinone, anthraquinone-2-carboxylic acid,1-chloroanthraquinone, 2-methyl-anthraquinone, and 1-5dihydroxyanthraquinone, 2-chloroanthraquinone.

Other compounds that can be used to control plant, animal, andmicroorganism growth either alone or in combination with each other andthe quinones, naphthalenediones, and anthraquinones noted above include9,10-dihydro-9-oxoanthracene (anthrone), 6′-methoxycinchonan-9-ol(quinine), 4-hydroxy-3-(3-oxo-1-phenyl butyl) -2H-1-benzopyran-2-one(warfarin), 2H-1-benzopyran-2-one (coumarin),7-hydroxy-4-methylcoumarin, 4-hydroxy-6-methylcoumarin,2[5-(4-aminophenoxy)pentyl]-1isoindole 1,3-(2H)-dione (amphotalide),sodium rhdixonate, 2-phenyl-1,3-indandione (phenindione), 2,5dihydroxy-3-undecyl-2,5 cyclohexadiene, spirulosin and thymoquinone.

Compounds that are particularly effective in controllingmacroinvertebrates include 2,3-methoxy-5-methyl-1,4-benzoquinone,2-methyl-1,4-naphthalenedione, 2-methyl-5-hydroxy-1,4-naphthalenedione,2-methyl -2-sodium metabisulfite-1,4-naphthalenedione,3-methyl-1,8-dihydroxyanthraquinone, 2-methyl-anthraquinone,1,2-dihydroxyanthraquinone, 1,4-naphthalenedione, and mixtures thereof.These compounds are also effective in controlling the growth ofdinoflagellates.

In one embodiment of the invention, mollusks, dinoflagellates, toxicbacteria, and algae are treated to inhibit growth by applying aneffective amount of compound selected from the group consisting of,2,3-methoxy-5-methyl-1,4-benzoquinone, 2-methyl-1,4-naphthalenedione,and mixtures thereof.

One preferred embodiment of the invention is directed to a method ofkilling or inhibiting the growth of mollusks, dinoflagellates, toxicbacteria, and/or algae by exposing the mollusks, dinoflagellates, toxicbacteria, and/or algae to an effective amount of a quinone,anthraquinone, naphthalenedione, or mixture thereof. The method iseffective in inhibiting the growth of toxic bacteria andmussels—particularly zebra mussels, and zebra mussel larvae, as well asother bivalves—by applying the aquacide compound to the water in aneffective amount. In a preferred embodiment, mussels, and particularlyzebra mussels and zebra mussel larvae, are treated to kill or inhibittheir growth by exposing the zebra mussels to a toxic amount of amolluskocide compound selected from the group consisting of2,3-methoxy-5-methyl-1,4-benzoquinone,2-methyl-5-hydroxy-1,4-naphthalenedione, 2-methyl-1,4-naphthalenedione,2-methyl-2-sodium metabisulfite-1,4-naphthalenedione,3-methyl-1,8-dihydroxyanthraquinone, 2-methylanthraquinone, and mixturesthereof.

In a further embodiment, these aquacidal compounds are incorporated asan active compound into a solid or liquid bait for agricultural use tokill or inhibit the growth of snails and slugs. The bait can be astandard bait as known in the art. In other embodiments, the aquacidalcompound is formed into a solution or dispersion and applied directly tothe plant in an effective amount to treat the plant for controllingsnails and slugs.

Aquacidal Amount

The amount of the aquacidal ingredient to be added will depend, in part,on the particular compound and the species of plant or animal beingtreated. As used herein, the term “effective amount” or “aquacidal”refers to an amount that is able to kill the target species or renderthe target specie population inert and otherwise not viable of sustainedvitality.

The method for treating water to kill a target plant or animalintroduces the aquacidal compound to the water in the amount of lessthan 1 wt %. Preferably, the aquacidal compound is added in an amountwithin the range of about 100 ppb to about 500 ppm (parts per million),more preferably in an amount within the range from about 500 ppb toabout 300 ppm, most preferably within the range of 500 ppb to 250 ppm,and especially in an amount within the range of 1 ppm to about 250 ppm.Generally, the amount of the aquacidal compound used in treatment ofballast tank water will range from about 1 ppm to about 200 ppm.

The target pest population should be exposed to the aquacide at theselected concentration for a time sufficient to kill the targetpopulation. Exposure periods sufficient are generally within the rangeof a at least one hour to a period of less than 96 hours (4 days) forboth fresh water as well as salt water. A preferred exposure is withinthe range from about two hours to about 48 hours. Routine sampling andtesting can be used to determine precise concentrations and exposuredurations for a specific aquacidal compound, water type, targetpopulation, method of introduction, and temperature.

Coatings

The aquacidal compounds of the invention can also be added to paints andcoatings in a concentration sufficient to provide population controlwithout adversely affecting the efficacy of the coating. The paint orcoating composition can be applied to a surface, such as the hull of aboat, intake pipes, ship chests, anchors, and other underwaterstructures to prevent the plants and animals from growing and adheringto the surface.

The paint or coating composition can be conventional marine paintcontaining various polymers or polymer-forming components. Examples ofsuitable components including acrylic esters, such as ethyl acrylate andbutyl acrylate, and methacrylic esters, such as methyl methacrylate andethyl methacrylate. Other suitable components include 2-hydroxyethylmethacrylate and dimethylaminoethyl methacrylate that can becopolymerized with another vinyl monomer, such as styrene. The paintcontains an effective amount of at least one aquacidal compound toinhibit plant an animal growth on a painted substrate. In embodiments ofthe invention, the aquacidal compound is included in an amount toprovide a concentration of the aquacidal compound at the surface of thecoating of at least 500 ppb, preferably about 1 ppm to 50 wt %, and morepreferably within the range of 100-500 ppm to provide a plant and animalcontrolling amount of the aquacide compound in the coating.

EXAMPLES

The effectiveness and toxicity levels of the compounds were evaluatedusing active plant and animal species. The various compounds were addedto the water at controlled rates and amounts. The results were observedand are recorded in Table 1 below.

The compounds were tested for efficacy on various plant and animalspecies according to the following protocols.

(a) Zebra Mussels (Larvae and Adults)

Zebra mussel broodstock were maintained in natural well water withcalcium and magnesium adjusted to a hardness level equivalent toapproximately 25 mg/l hardness.

At 20° C., larvae remain in the free-swimming state for 30-40 days priorto settlement. Bioassays using early larval stages of this species arevariants on standard oyster embryo bioassays. Assays are conducted atthe embryo, trochophore and D-hinge stage.

The assays examined the toxicity of various quinones to the earliestlife history stages, namely embryo to trochophore stage (2-17 hours);trochophore stage (2-17 hours); trochophore to D-hinge stage (17-48hours); and embryo to D-hinge stage (2-48 hours).

Approximately 25 adults from broodstock (held at 10-12° C.) were cleanedof debris and transferred to 1500 ml glass beakers containingapproximately 800 ml of culture water. Water temperature was rapidlyraised to 30-32° C. by the addition of warm water. Mussels treated thisway usually spawn within 30 minutes. If no spawning occurred within thistime, a slurry made from ripe gonads homogenized in culture water isadded.

A successful spawn yielded >50,000 eggs/female. To check for successfulfertilization, zygotes were transferred to a Sedgewick-Rafter cell forcounting and examination under a binocular microscope. Fertilized eggswere seen to be actively dividing and reached the 8-cell stage between2-3 hours following fertilization. A better than 70% fertilization rateis considered indicative of viable experimental material.

Assays were conducted on at least 500 embryos/larvae in each of 4replicates. A range of 5 test concentrations (in the ppm range) pluscontrols were used. A density of 10 embryos per ml were used for embryoassays, and for D-hinge larvae 2 larvae/ml were used. The tests werestatic non-renewal. Any assay lasting 24 hours or longer received food(cultured Neochloris @ 5×104 cells ml-l) at 24 hour intervals.

Following counting and adjustment of densities, embryo assays werestarted as early as 2 hours following fertilization by inoculating aknown number of embryos into the test media. Late stages were held inculture water until inoculation. Survivors were counted usingSedgewick-Rafter cells, with adjustments for control mortality usingAbbott's formula. Probit and Dunnett's test are used to obtain the LD50,Lowest Observed Effect Concentration (LOEC) and No Observed EffectConcentration (NOEC) (Toxcalc 5.0).

(b) Fathead Minnow Acute Assay (Fish assay)

Fathead minnows (Pimephales promelas) from in-house laboratory cultureswere used for these tests. Animals were cultured in natural well waterwith hardness adjusted to >50 ppm (CaCO₃) equivalents. Fish were spawnedin a 20 gal spawning tank containing PVC tubing as refuges. Newlyhatched larvae were transferred to a holding tank at densities of50-100/1 until use. Brine shrimp nauplii (Artemia) were used as food.

The tests were static renewal. The test durations were 48 hours and 96hours. The temperature was 20° C. ±1° C. Light quality was ambientlaboratory illumination. Light intensity was 10-20 E/m²/sec (50-100ft-c). The photoperiod was 16 hours of light and 8 hours of dark. Thetest container was 400 ml. Renewal of test solutions occurred at 48hours. The age of test organisms was 1-14 days, with a 24 hour agerange. There were 10 organisms per container. There were 3 replicatesper concentration of individual quinones in the ppm range. There are 5test concentrations plus controls (initial range-finding tests performedon logarithmic series). All tests were conducted within 5 hours ofdissolving the test compound. Animals were fed Artemia nauplii prior tothe test and 2 hours prior to the 48 hour test solution renewal. Oxygenlevels were maintained at >4.0 mg/L. Natural well water adjusted to >50mg/L hardness equivalents was used for dilution.

The test objectives are to determine LC50,LOEC and NOEC. The testacceptability threshold is 90% or greater survival in controls. Data areanalyzed using Toxcalc 5.0.

(c) Dinoflagellate (Prorocentrum Minimum) Assay

The dinoflagellate prorocentrum minimum was cultured at the ChesapeakeBiological Laboratory culture facility from in-house stocks grown up asa 1 liter culture in sterilized 16 ppt salinity filtered water fortifiedwith f/2 nutrient media. The culture was diluted to 5 liters withfiltered estuarine water 16 ppt salinity prior to the experiments. Theapproximate starting cell density was 2×10⁶ cells per ml.

Each 600 ml glass beaker containing 400 ml dinoflagellate culture wasallowed to grow under continuous fluorescent light following theexposure treatments. At daily intervals, samples were taken for cellcounting and microscopical examination, extraction of chlorophyllpigments with acetone and for direct in-vivo chlorophyll fluorescencedetermination.

100 ml of each dinoflagellate culture treatment in triplicate werefiltered through a 25 mm GFF filter under gentle vacuum. The filterswere folded and placed in polypropylene centrifuge tubes and exactly 4ml of HPLC grade acetone added. The samples were sonicated with a probe(Virsonic 50) for approximately 2 minutes to disrupt cells after whichthey are allowed to extract at 4° C. overnight in a refrigerator. Aftercentrifuging for 5 minutes, the supernatant was transferred to a quartzfluorometer cell and the fluorescence recorded using a Hitachi F4500scanning fluorescence detector. Excitation was fixed at 436 nm with a 10nm slit and the emission is recorded at 660 nm with a 10 nm slit. Thephotomultiplier is operated at 700 V. Authentic chlorophyll a and b(Sigman Chemicals) were dissolved in HPLC grade acetone to calibrate thespectrofluorometer. Three point calibrations were performed intriplicate on a daily basis and relative fluorescence response convertedinto units of ug/l.

In-vivo fluorimetry with the Hitachi F4500 involves suspending the algalcells and transferring an aliquot to a disposable polycarbonate cuvetteand recording the emission spectra from 600-720 nm with excitation fixedat 436 nm with a 10 nm slit width.

Direct cell counts were made with a compound binocular microscope and ahemacytometer counting triplicate samples in 80 squares.

End-points for quinone toxicity include cell motility, inhibition ofcell division, inhibition of chlorophyll synthesis and chloroplatebleaching.

(d) Chlorella Assay

Assays for other species of phytoplankton including Chlorella sp. andIsochrysis galbana followed the above outlined procedures.

(e) Copepod Assays (Eurytemora Affinis)

Cultures of Eurytemora affinis were continuously maintained in 15seawater in a 8/16 hours light/dark regime fed every 48 hours onIsochrysis galbana. Toxicity bioassays are conducted on early instarnaupliar larvae (chronic mortality/fecundity assay) or adults (acuteLC50 assay).

Larvae were collected as follows. Cultures were filtered with a 200 mNitex filter to separate the adults from earlier stages. Adults werethen allowed to spawn for 48-72 hours in order to produce stage 1-3naupliar larvae to be used for the assay. Assays were conducted onbatches of 10 larvae per treatment (in triplicate). At 20° C., assayswere continued for 12 days (shorter at higher temperatures). Endpointswere the percentage of F0 generation (present as adults) and totalnumbers of F1 generation (present as eggs or naupliar larvae). LC50assays on adult copepods were conducted for 24 or 48 hours withpercentage mortality as the end-point. All assays were conducted at 15salinity on a 8 hour/16 hour light/dark regime.

(f) Dinoflagellate Cysts (Glenodinium sp.)

Dinoflagellate cysts were collected from marine sediments cleaned ofdebris using mild ultrasonic cleansing and exposed to ppm levels ofvariety of quinones. Light microscopy and epifluorescence microscopywere employed to examine the cysts for oxidative damage and chloroplastdisruption following treatment at the ppm level.

TABLE 1 Ex. IUPAC Nomenclature Empirical Formula Organism Toxicity Data(1) 2-methyl-5-hydroxy-1,4- C₁₁H₈O₃ T. isochrysis Toxic at 50 ppbnapthoquinone galbana Neochloris Toxic at 500 ppm Zebra larvae Toxic at200 ppb E. affinis 5 ppm < 10 min Artemia Toxic at 5 ppm salina Fisheggs Kills & hatch prevention @ 1 ppm Minnow Toxic at 1 ppm larvae (2)2-methyl-1,4- C₁₁H₈O₂ T. isochrysis Toxic at 500 ppb naphthalenedionegalbana (Vitamin K₃) Zebra mussel Toxic at 500 ppm larvae Oyster larvae1 ppm E. affinis 5 ppm < 15 min Artemia Toxic at 5 ppm salina Fish eggsKills & hatch prevention @ 1 ppm (3) 2-methyl-2-sodium C₁₁H₁₀SO₅Na T.isochrysis Toxic at 500 ppb metabisulfite-1,4- galbana naphthalenedioneZebra larvae Toxic at 1 ppm Oyster larvae 500 ppb E. affinis 5 ppm < 15min Artemia Toxic at 5 ppm salina Fish eggs Kills & hatch prevention @ 1ppm (4) Anthrone C₁₄H₁₀O T. isochrysis Toxic at 2 ppm galbana (5) 1,2-C₁₄H₈O₄ T. isochrysis Toxic at 1 ppm dihydroxyanthraquinone galbana E.affinis Toxic at 1 ppm Artemia Toxic at 5 ppm salina (6) 3-methyl-1,8-C₁₅H₁₀O₄ T. isochrysis Toxic at 1 ppm dihydroxyanthraquinone galbanaZebra mussel Toxic at 1 ppm larvae (7) anthraquinone-2- C₁₅H₈O₄ T.isochrysis Toxic at 1 ppm carboxylic acid galbana E. affinis 5 ppm < 5hours (8) 1-chloroanthraquinone C₁₄H₇O₂ T. isochrysis Toxic at 500 ppbgalbana Neochloris Toxic at 500 ppb E. affinis 5 ppm < 5 hours (9)2-methylanthraquinone C₁₅H₁₀O₂ T. isochrysis Toxic at 500 ppb galbanaNeochloris Toxic 1 ppm Zebra larvae Toxic at 200 ppm E. affinis 5 ppm <45 min Artemia Toxic at 5 ppm salina (10) 1,4-naphthalenedione C₁₀H₆O₂T. isochrysis Toxic at 1 ppm galbana Oyster larvae Toxic at 5 ppm E.affinis 5 ppm < 10 min (11) anthraquinone C₁₄H₈O₂ E. affinis 5 ppm < 4hours (12) 1,4-benzoquinone C₆H₄O₂ T. isochrysis Toxic at 500 ppbgalbana Fish eggs 50% mortality at 5 ppm. Control hatch at 1 ppm (13)methyl-1,4-benzoquinone C₇H₆O₂ T. isochrysis Toxic at 500 ppb(toluquinone) galbana (14) 2,3-methoxy-5-methyl- C₉H₁₀O₄ T. isochrysisToxic at 5 ppm 1,4-benzoquinone galbana

Example 15

Banana snails (Bulimulis altemata) were obtained from a commercialsupplier and were fed lettuce leaves until the start of the bioassay.

Ten snails were placed in covered 1 liter glass beakers, onapproximately 50 cm² lettuce leaves which had been sprayed with a finemist of an aqueous solution of 2,3-methoxy-5-methyl-1,4-benzoquinone atthree concentrations: 5, 10 and 20 mg/l. The treated leaves were allowedto dry before exposure to the snails. 10 snails were placed onapproximately 50 cm² of untreated lettuce leaf as a control. Treatmentsand controls were maintained at approximately 20° C. in the dark. Theywere observed at 24 and 48 hours for signs of mortality and feedingactivity.

In all treatments, the snails demonstrated significant avoidancerelative to control. Several snails of the treatment group withdrew intotheir shells and exhibited no feeding activity at all (leaves werecompletely intact). Others climbed up the walls of the beakers away fromthe leaves. This avoidance behavior was again observed after 48 hours.In contrast, the control group of snails consumed more than 10% of theleaf surface area after 24 hours and continued to feed and had consumedabout 20% of the leaf after 48 hours.

While various embodiments have been selected to illustrate theinvention, it will be understood to those skilled in the art thatvarious changes and modifications can be made to the process disclosedherein without departing from the spirit and scope of the invention asdefined in the appended claims.

What is claimed is:
 1. A method for controlling a population of targetaquatic pest microorganisms by applying an aquacidal compound to waterinfected with said target aquatic pest microorganisms, wherein saidaquacidal compound comprises at least one anthraquinone and is appliedin an amount that is effective to kill the target aquatic pestpopulation.
 2. The method of claim 1, wherein said population of targetaquatic pest microorganisms is selected from the group consisting ofviruses, protists, fungi, molds, plants, holoplanktonic organisms,meroplanktonic organisms, demersal organisms, benthic organisms,detached or floating biota, bacteria whether or not encysted,protozoans, algae, pyrrophyta, cryptophyta, chrysophyta, porifera,platyhelminthes, pseudocoelomates, annelid worms, zebra mollusks,bivalves, larval forms of copepods, ostracods, mysids, gammarids, larvalforms of decapods, and larval teleost fish.
 3. The method of claim 1,wherein said population of target pest microorganisms is selected fromthe group consisting of viruses, protists, holoplanktonic organisms, andmeroplanktonic organisms.
 4. The method of claim 1 wherein saidpopulation of target pest microrganisms is selected from the groupconsisting of demersal organisms, benthic organisms, detached orfloating biota, bacteria whether or not encysted, and protozoans.
 5. Themethod of claim 1 wherein said population of target pest microrganismsis selected from the group consisting of algae, pyrrophyta, cryptophyta,chrysophyta, porifera, platyhelminthes, pseudocoelomates, annelid worms,zebra mussels, bivalves, larval forms of copepods, ostracods, mysids,gammarids, larval forms of decapods, and larval teleost fish.
 6. Themethod of claim 1 wherein said population of target pest microorganismsis selected from the group consisting of spiny water flea and bacteria.7. A method according to claim 1 wherein said target aquatic pest isselected from the group consisting of bacteria whether or not encysted,protozoans, algae, dinoflagellates, dinoflagellate cysts, zebra mussels,and zebra mussel larvae.
 8. A method according to claim 1 wherein saidtarget aquatic pest is selected from the group consisting of bacteria,algae, dinoflagellates, dinoflagellate cysts, zebra mussels, and zebramussel larvae.
 9. A method according to claim 1 wherein said targetaquatic pest is a bacteria.
 10. A method according to claim 1 whereinsaid bacteria is a Vibrio species.
 11. A method according to claim 1wherein said target aquatic pest is a zebra mussel or zebra mussellarvae.
 12. A method according to claim 1 wherein said target aquaticpest is a demersal organism.
 13. A method according to claim 1 whereinsaid target aquatic pest is a benthic organism.
 14. A method accordingto claim 1 wherein said target microorganism is a dinoflagellate cyst.15. The method of claim 1, wherein said aquacidal compound has theformula:

wherein R₁ is hydrogen, hydroxy, chloro; R₂ is hydrogen, methyl, chloro,hydroxy, carbonyl, or carboxyl group; R₃ is hydrogen or methyl group; R₄is hydrogen; R₅ is hydrogen or hydroxyl group; R₆ and R₇ are hydrogen;and R₈ is hydrogen or hydroxyl group.
 16. The method of claim 1, whereinsaid aquacidal compound is selected from the group consisting of 9,10anthraquinone, 1,2-dihydroxyanthraquinone (alizarin),3-methyl-1,8-dihydroxyanthraquinone, anthraquinone-2-carboxylic acid,1-chloroanthraquinone, 2-methyl-anthraquinone, and 1-5dihydroxyanthraquinone, 2-chloroanthraquinone.
 17. The method of claim 1wherein said aquacidal compound is present in an amount of less than 1wt %.
 18. The method of claim 1 wherein said aquacidal compound ispresent in an amount within the range of 100 ppb to 500 ppm.
 19. Themethod of claim 1 wherein said aquacidal compound is present in anamount within the range of 500 ppb to 300 ppm.
 20. The method of claim 1wherein said aquacidal compound is present in an amount within the rangeof 1 ppm to 200 ppm.
 21. The method of claim 1 wherein the target pestpopulation is exposed to said aquacidal compound for at least one hourfollowing application of said aquacidal compound.
 22. The method ofclaim 21 wherein said target pest population is exposed to saidaquacidal compound for 1-96 hours.
 23. The method of claim 22 whereinsaid target pest population is exposed to said aquacidal compound for2-48 hrs.
 24. The method of claim 1 wherein said population of targetpest microorganisms are located in a ballast water reservoir.
 25. Themethod of claim 1 wherein said population is Vibrio Cholera or VibrioFisheri.
 26. A method for killing a target population of mollusk pestsin an aqueous system hosting said population comprising the step of:adding to said aqueous system an amount of an aquacidal compound that issufficient to kill said target population, said aquacidal compoundhaving the formula:

wherein R₁ is hydrogen, hydroxy, chloro; R₂ is hydrogen, methyl, chloro,hydroxy, carbonyl, or carboxyl group; R₃ is hydrogen or methyl group; R₄is hydrogen; R₅ is hydrogen or hydroxyl group; R₆ and R₇ are hydrogen;and R₈ is hydrogen or hydroxyl group.
 27. The method of claim 26,wherein said mollusk pests are selected from the group consisting ofmussels, clams and snails.
 28. The method of claim 26, wherein saidmollusk pests are selected from the group consisting of zebra musselsand Asiatic clams.
 29. The method of claim 26 wherein said mollusk pestsare exposed to said aquacidal compound for a period of time sufficientto kill said pests.
 30. The method of claim 26 wherein said molluskpests are exposed to said aquacidal compound for a period of time withinthe range of 1-96 hours and at a concentration within the range of 100ppb to 500 ppm.